The actions of ethanol on the central nervous system involve a number of biochemical sites. The ligand-gated ion channels, including the serotonin-3 (5-HT3) receptor, have recently been the focus of significant research interest. Substantial behavioral data suggest that ethanol's reinforcing stimulus effects may be mediated at least in part through its interaction with 5-HT3 receptors. A number of 5-HT3-R antagonists antagonize ethanol consumption in rats and clinical studies suggest that these compounds may show promise in decreasing alcohol drinking in humans. There is also abundant electrophysiological evidence demonstrating that the function of homeric 5-HT3 receptors is potentiated by pharmacologically-relevant concentrations of ethanol. The work proposed in this project involves the study of the interactions of ethanol with 5-HT3 receptors on the molecular level. The overall objective of the work is the identification of specific amino acid residues of the 5-HT3 receptor that mediate the enhancing effects of ethanol of 5-HT3 receptor function. Our secondary goal is to produce a mutated 5-HT3 receptor that will behave, as much as possible, like a wild-type receptor except that it will be largely or completely insensitive to the enhancing effects of ethanol. We believe that the receptor's sensitivity to ethanol can be selectively eliminated without altering other parameters of 5-HT3 receptor function, such as serotonin sensitivity or desensitization rates. Our recent success in identifying amino acid residues responsible for ethanol enhancement of the function of the related GABAA and glycine receptors give us confidence that we will be able to construct ethanol-insensitive 5-HT3 receptors that we will test in vitro using the Xenopus oocyte expression system. Our long term goal is the production of genetically engineered mice expressing 5- HT3 receptors that are in all ways normal except that the receptors are either completely or largely insensitive to ethanol. Comparisons of these animals with wild-type controls, by members of this Center, will help to delineate the role of the 5-HT3 receptor in the behavioral effects of ethanol. An understanding of the mechanisms of ethanol actions will aid in the development of rational therapies for alcohol abuse and alcoholism.